CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. ERC_2015_StG _ 680156 _ZPR). J.B. acknowledges Fundação para a Ciência e a Tecnologia (FCT) for an FCT Scientific Stimulus Grant ( CEECIND/03482/2018 ). J.A. is a PhD fellow from FCT ( SFRH/BD/145110/2019 ). R.B.C. was funded by FCT ( ON2201403-CTO-BPD ) and EMBO (Short-Term Fellowship). We thank Joaquin Letelier, Ana Novoa, and Moises Mallo for important advice establishing the current protocol. We also thank Joana Marques for constant assistance in establishing the protocol

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs ß cell differentiation, compatible with the antagonistic role of Bmp in ß cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs ß cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.We thank José Luís Gómez-Skarmeta and Paulo Pereira for helpful suggestions and critical reading of the manuscript, Vitor Silva for support in the phylogenetic tree design, and Marta Duque for help in designing the graphical abstract. This study was supported by the European Research Council ( ERC ) under the European Union’s Horizon 2020 research and innovation program (grant ERC2015StG680156ZPR ). J.B. acknowledges Fundação para a Ciência e a Tecnologia ( FCT ) for an FCT Investigator position (grant IF/00654/2013 ). J.T. and J.P.A. are PhD fellows from FCT (grant SFRH/BD/126467/2016 to J.T. and grant SFRH/BD/145110/2019 to J.P.A.). M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation program (grant 668981 ). The authors acknowledge the support of i3S Scientific Platform Advanced Light Microscopy, a member of the national infrastructure PPBI ( Portuguese Platform of BioImaging ) (supported by POCI010145FEDER022122 ). We acknowledge the I3S hpc facility, used for processing the 4C-seq data, and André Torres for the useful support. We also thank Francesco Argenton for providing us with a vector for generating an insulin-targeting probe. Finally, we thank Yolanda Roncero and Isabel Guedes for helping to screen and maintain the nog2 ED301 zebrafish line and Tania Medeiros for preliminary work on the function of the notochord in pancreas development

Quantification of epigenetic and genetic 2nd hits in CDH1 during hereditary diffuse gastric cancer syndrome progression

BACKGROUND & AIMS: Hereditary diffuse gastric cancer (HDGC) families carry CDH1 heterozygous germline mutations; their tumors acquire complete CDH1 inactivation through "2nd-hit" mechanisms. Most frequently, this occurs via promoter hypermethylation (epigenetic modification), and less frequently via CDH1 mutations and loss of heterozygosity (LOH). We quantified the different 2nd hits in CDH1 occurring in neoplastic lesions from HDGC patients. METHODS: Samples were collected from 16 primary tumors and 12 metastases from 17 patients among 15 HDGC families; CDH1 mutations, LOH, and promoter hypermethylation were analyzed. E-cadherin protein expression and localization were determined by immunohistochemistry. RESULTS: Somatic CDH1 epigenetic and genetic alterations were detected in lesions from 80% of HDGC families and in 75% of all lesions analyzed (21/28). Of the 28 neoplastic lesions analyzed, promoter hypermethylation was found in 32.1%, LOH in 25%, both alterations in 17.9%, and no alterations in 25%. Half of the CDH1 2nd hits in primary tumors were epigenetic modifications, whereas a significantly greater percentage of 2nd hits in metastases were LOH (58.3%; P = .0274). Different neoplastic lesions from the same patient frequently displayed distinct 2nd-hit mechanisms. Different 2nd-hit mechanisms were also detected in the same tumor sample. CONCLUSION: The 2nd hit in CDH1 frequently occurs via epigenetic changes in HDGC primary tumors and LOH in metastases. Because of the concomitance and heterogeneity of these alterations in neoplastic lesions and the plasticity of hypermethylated promoters during tumor initiation and progression, drugs targeting only epigenetic alterations might not be effective, particularly in patients with metastatic HDGC

The UPF1 RNA surveillance gene is commonly mutated in pancreatic adenosquamous carcinoma

Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are the first known unique molecular signatures of ASC